The invention relates to short integuments1 nucleic acids and proteins, and to plants having altered phenotypes when transformed with short integumentsl nucleic acids.
According to recent estimates, the global demand for crop plants such as rice, wheat, and maize should increase by 40% by 2020. It is thought that classical plant breeding technology, which led to the green revolution in the late 1960s, will contribute less and less to meet this increasing demand, whereas plant genetic engineering will contribute increasingly more. An important thrust area in plant genetic engineering is the identification and use of genes implicated in asexual production of seeds, or xe2x80x9capomixis.xe2x80x9d Apomixis is thought to be an agronomically desirable trait that should enable seed companies and farmers to lock-in a favorable combination of genes for maximum grain yield without having to lose the gene combination in the next sexual generation. Genes for apomixis have not yet been identified. It is thought that genes that are generally important for very early embryo/seed development may be important for apomixis. A second important thrust is the production of early flowering varieties of plants such that breeding time can be reduced.
The evolution of flowering plants may have entailed a modification of primitive leaf or leaf-like structures that contained naked ovules on their surfaces, to specify floral organs that ultimately evolved to surround the ovules (Herr, xe2x80x9cThe Origin of the Ovule,xe2x80x9d Am. J. Bot. 82:547-564 (1995); Stebbins, Flowering Plants: Evolution Above the Species Level, Cambridge, Mass.: Harvard University Press, pp. 199-245). This view of angiosperm evolution predicts that the genetic regulatory network that controls ovule development should be interlaced with that which triggers flowering. Ovule, as the precursor of seed, is the link to the next generation. Genetic regulatory pathways that are important for early vegetative development of the embryo inside the ovule, for late reproductive development leading to the production of ovules, and for morphogenesis of the haploid female gametophyte, are crucial areas of investigation which can lead to enhanced agricultural practices.
Several genes important for ovule development have been identified in Arabidopsis thaliana (Reiser et al., xe2x80x9cThe Ovule and the Embryo Sac,xe2x80x9d The Plant Cell 5:1291-1301 (1993)). BELL1, a so-called cadastral gene that encodes a homeodomain protein (Reiser et al., xe2x80x9cThe BELL1 Gene Encodes a Homeodomain Protein Involved in Pattern Formation in the Arabidopsis Ovule Primordium,xe2x80x9d Cell 83, 735-742 (1995)), controls the expression of the floral organ identity gene AG within the ovule and thereby controls morphogenesis of ovule integuments (Modrusan et al., xe2x80x9cHomeotic Transformation of Ovules into Carpel-Like Structures in Arabidopsis,xe2x80x9d The Plant Cell 6:333-349 (1994); Ray et al., xe2x80x9cThe Arabidopsis Floral Homeotic Gene BELL (BEL1) Controls Ovule Development Through Negative Regulation of AGAMOUS (AG) Gene,xe2x80x9d Proc. Natl. Acad. Sci. USA 91:5761-5765 (1994)). SUPERMAN, another cadastral gene that restricts the spatial expression pattern of the floral organ identity gene AP3 (Sakai et al., xe2x80x9cRole of SUPERMAN in Maintaining Arabidopsis Floral Whorl Boundaries,xe2x80x9d Nature 378:199-203 (1995)), is important in ovule integument development (Gaiser et al., xe2x80x9cThe Arabidopsis SUPERMAN Gene Mediates Asymmetric Growth of the Outer Integument of Ovules,xe2x80x9d The Plant Cell 7:333-345 (1995)). The organ identity gene AP2 is also known to control ovule morphogenesis (Modrusan et al., xe2x80x9cHomeotic Transformation of Ovules into Carpel-Like Structures in Arabidopsis,xe2x80x9d The Plant Cell 6:333-349 (1994)). By contrast, no known meristem identity or flowering control gene had, until now, been demonstrated to have a role in ovule development.
A gene termed SHORT INTEGUMENTS1 (SIN1), genetically detected in the model plant Arabidopsis thaliana by mutational studies has been determined to be an important regulatory gene for plant reproductive development. The SIN1 gene is required for normal ovule development (Lang et al., xe2x80x9csin1, A Mutation Affecting Female Fertility in Arabidopsis, Interacts with mod1, its Recessive Modifier,xe2x80x9d Genetics 137:1101-1110 (1994); Reiser et al., xe2x80x9cThe Ovule and the Embryo Sac,xe2x80x9d The Plant Cell 5:1291-1301 (1993); Robinson-Beers et al., xe2x80x9cOvule Development in Wild-Type Arabidopsis and Two Female Sterile Mutants,xe2x80x9d Plant Cell 4:1237-1250 (1992)). The original isolate of the sin1 mutation (sin1-1 allele) was identified as one causing a female sterile phenotype (Robinson-Beers et al., xe2x80x9cOvule Development in Wild-Type Arabidopsis and Two Female Sterile Mutants,xe2x80x9d Plant Cell 4:1237-1250 (1992)). Ovules of the original isolate have short integuments and a defective megagametophyte (see Reiser et al., xe2x80x9cThe Ovule and the Embryo Sac,xe2x80x9d The Plant Cell 5: 1291-1301 (1993)) for a review on ovule structure; Baker et al., xe2x80x9cInteractions Among Genes Regulating Ovule Development in Arabidopsis thaliana,xe2x80x9d Genetics 145:1109-1124 (1997), for a recent genetic analysis; Schneitz et al., xe2x80x9cDissection of Sexual Organ Ontogenesis: A Genetic Analysis of Ovule Development in Arabidopsis thaliana,xe2x80x9d Development 124:1367-1376 (1997), for a summary of the known mutants affected in ovule development). It has been shown that the originally-described Sin1xe2x88x92 mutant phenotype is a result of an interaction between sin1, and mod1, its recessive modifier (Lang et al., xe2x80x9csin1, A Mutation Affecting Female Fertility in Arabidopsis, Interacts with mod1, Its Recessive Modifier,xe2x80x9d Genetics 137:1101-1110 (1994)), and that mod1 is erecta, a mutation in a putative serine-threonine receptor protein kinase gene. The sin1-1 or sin1-2 mutation acting alone causes a defect in the coordination of growth of the two sheets of cells of the inner and outer integuments. All other originally described effects on the ovule, such as the lack of outer integument cell expansion and arrest of the megagametophyte, are due to secondary genetic interactions with erecta. There are several prospective protein phosphorylation sites within the SIN1 protein, and these might be substrates of protein kinases, such as the ERECTA product (Torii et al., xe2x80x9cThe Arabidopsis ERECTA Gene Encodes a Putative Protein Kinase with Extracellular Leucine-Rich Repeats,xe2x80x9d Plant Cell 8:735-746 (1996)).
In plants homozygous for the weaker sin1-2 mutant allele, approximately 40% of all ovules in any flower mature into seeds. But these seeds frequently contain embryos arrested at different stages of development, some of which germinate to produce abnormal seedlings. Genetic analysis shows that the maternal expression of the SIN1 gene is necessary for embryo development (Ray et al., xe2x80x9cMaternal Effects of the Short Integument Mutation on Embryo Development in Arabidopsis,xe2x80x9d Dev. Biol. 180:365-369 (1996)).
Not only does this gene function in the formation of seeds, SIN1 is the only identified plant gene whose maternal expression is important for pattern formation in the zygotic embryo (Ray et al., xe2x80x9cMaternal Effects of the Short Integument Mutation on Embryo Development in Arabidopsis,xe2x80x9d Dev. Biol. 180:365-369 (1996)). Both sin1-1 and sin1-2 alleles have the maternal-effect embryonic lethality phenotype (Ray et al., xe2x80x9cMaternal Effects of the Short Integument Mutation on Embryo Development in Arabidopsis,xe2x80x9d Dev. Biol. 180:365-369 (1996)). The wild type SIN1 allele when transmitted through the pollen is unable to rescue the deleterious effects on embryogenesis of a homozygous maternal sin1-2 mutation. Ray et al. have shown that a wild type allele of SIN1 in the endosperm cannot rescue the maternal-effect of sin1-2 (Ray et al., xe2x80x9cMaternal Effects of the Short Integument Mutation on Embryo Development in Arabidopsis,xe2x80x9d Dev. Biol. 180:365-369 (1996)). This is the first demonstration of a maternal effect embryonic pattern formation gene in a plant.
In Arabidopsis thaliana, meristem development progresses through at least three distinct phases: from vegetative (V) through inflorescence (I) to the floral (F) mode, a process known as the xe2x80x9cVxe2x86x92Ixe2x86x92F switch.xe2x80x9d It has been shown that the sin1 mutation causes a defect in the Vxe2x86x92Ixe2x86x92F switch. SIN1 is needed for the expression of the early flowering phenotype imparted by a TERMINAL FLOWER1 (tfl1) mutation, and tfl1 sin1 double mutants do not produce pollen. Furthermore, sin1-1 allele enhances the effect of an APETALA1 (ap1) mutation. Thus, SIN1 represents a genetic connection between ovule development and control of flowering.
In addition, the function of SIN1 gene is important for controlling the time to flower, another important agronomic factor because the timing of seed production depends on the flowering time. Ray et al. have shown by genetic analysis that SIN1 gene regulates the activity of a master switch gene, LEAFY (LFY) that controls flowering time in Arabidopsis thaliana. The LEAFY gene from Arabidopsis thaliana was shown to accelerate the flowering time of aspen (an economically important timber plant) from many years to a few months. Additionally, sin1 mutants are late flowering (Ray et al., xe2x80x9cSHORT INTEGUEMNT (sin1), A Gene Required For Ovule Development in Arabidopsis, Also Controls Flowering Time,xe2x80x9d Development 122, 2631-2638 (1996)) due to the production of an excess of vegetative leaves and lateral inflorescence axes before producing the floral primordia, which suggests a role of SIN1 in meristem fate determination. The ability to improve crop plant production through genetic engineering requires the identification and manipulation of previously unidentified genes that control developmentally important plant processes, including ovule development and flowering in plants.
The present invention is directed to overcoming the deficiencies in the prior art.
The present invention relates to an isolated nucleic acid molecule encoding a short integuments1 protein.
The present invention also relates to an isolated short integuments1 protein.
The present invention also relates to a method of regulating flowering in plants that involves transducing a plant with a DNA molecule encoding a short integuments1 protein under conditions effective to regulate flowering in the plant.
The present invention also relates to a method of increasing fertility in plants that involves transducing a plant with a DNA molecule encoding a short integuments1 protein under conditions effective to increase fertility.
The present invention also relates to a method of increasing fecundity in plants that involves transducing a plant with a DNA molecule encoding a short integuments 1 protein under conditions effective to increase fecundity.
The present invention also relates to a method of decreasing fertility in plants that involves transducing a plant with a DNA molecule encoding a short integuments1 protein mutated to cause disruption of the DNA molecule under conditions effective to decrease fertility.
The present invention also relates to an expression vector containing a DNA molecule encoding a short integuments1 protein, and plant cells, plant seeds and transgenic plants transformed with a DNA molecule encoding a short integuments1 protein.
It is expected that elucidation of post-transcriptional regulation in plants will contribute significantly to the ability to control plant production through biotechnology. However, very little is currently understood about mechanisms of post-transcriptional controls, especially in plant reproduction. This invention overcomes this and other deficiencies in the art, as the SIN1 gene and its encoded protein, which play a vital role in fertility, seed production and flowering time control in plants, provide the agronomist with important tools for engineering the expression of genes involved in seed/embryo development and flowering time.